Myricetin, rosmarinic and carnosic acids as superior natural antioxidant alternatives to α-tocopherol for the preservation of omega-3 oils

Article abstract of DOI:10.1016/j.foodchem.2016.06.038

22 natural polyphenols are compared to 7 synthetic antioxidants including BHT, BHA, TBHQ and PG with regard to their ability to protect omega-3 oils from autoxidation. The antioxidant efficiency of phenols is assessed using the DPPH test and the measurement of oxygen consumption during the autoxidation of oils rich in omega-3 fatty acids. Also, the bond dissociation enthalpies (BDE) of the Ar-OH bonds are calculated and excellent correlations between thermodynamic, kinetic and oxidation data are obtained. It is shown that kinetic rates of hydrogen transfer, number of radicals scavenged per antioxidant molecule, BDE and formation of antioxidant dimers from the primary radicals play an important role regarding the antioxidant activity of phenols. Based on this, it is finally shown that myricetin, rosmarinic and carnosic acids are more efficient than α-tocopherol and synthetic antioxidants for the preservation of omega-3 oils.

Full text of DOI:10.1016/j.foodchem.2016.06.038

Accepted Manuscript
Myricetin, rosmarinic and carnosic acids as superior natural antioxidant alter-
natives to α-tocopherol for the preservation of omega-3 oils
Romain Guitard, Jean-François Paul, Véronique Nardello-Rataj, Jean-Marie
Aubry
PII:
S0308-8146(16)30931-1
DOI:
http://dx.doi.org/10.1016/j.foodchem.2016.06.038
FOCH 19379
Reference:
Food Chemistry
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Revised Date:
Accepted Date:
13 January 2016
12 May 2016
14 June 2016
Please cite this article as: Guitard, R., Paul, J-F., Nardello-Rataj, V., Aubry, J-M., Myricetin, rosmarinic and carnosic
acids as superior natural antioxidant alternatives to α-tocopherol for the preservation of omega-3 oils, Food
Chemistry (2016), doi: http://dx.doi.org/10.1016/j.foodchem.2016.06.038
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Myricetin, rosmarinic and carnosic acids as superior natural antioxidant
alternatives to α-tocopherol for the preservation of omega-3 oils
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Romain Guitarda, Jean-François Paula, Véronique Nardello-Rataja* and Jean-Marie
Aubrya*
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Univ. Lille, CNRS, Centrale Lille, Univ. Artois, UMR 8181 – UCCS – Unité de Catalyse et
Chimie du Solide, F-59000 Lille, France
E-mail: jean.marie-aubry@univ-lille.fr and veronique.rataj@univ-lille1.fr
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Abstract
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22 natural polyphenols are compared to 7 synthetic antioxidants including BHT, BHA, TBHQ
and PG with regard to their ability to protect omega-3 oils from autoxidation. The antioxidant
efficiency of phenols is assessed using the DPPH• test and the measurement of oxygen
consumption during the autoxidation of oils rich in omega-3 fatty acids. Also, the bond
dissociation enthalpies (BDE) of the Ar-OH bonds are calculated and excellent correlations
between thermodynamic, kinetic and oxidation data are obtained. It is shown that kinetic rates
of hydrogen transfer, number of radicals scavenged per antioxidant molecule, BDE and
formation of antioxidant dimers from the primary radicals play an important role regarding
the antioxidant activity of phenols. Based on this, it is finally shown that myricetin,
rosmarinic and carnosic acids are more efficient than α-tocopherol and synthetic antioxidants
for the preservation of omega-3 oils.
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Keywords: Omega-3 FAMEs; Autoxidation; Natural phenolic antioxidants; DPPH; BDE;
Linseed oil
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1. Introduction
Omega-3 essential fatty acids have drawn the attention of scientists for many years
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and studies have multiplied in recent decades, highlighting their virtues and indispensable
character to the proper functioning of human bodies. The discovery of the positive effects of
omega-3 fatty acids has been the result of contributions from several scientists all over the
world who have made some unexplained observations (Dyerberg, Bang, & Hjoerne, 1975).
The main omega-3 fatty acids found in animal and plant food are α-linolenic acid (ALA,
18:3), eicosapentaenoic acid (EPA, 20:5) and docosahexaenoic acid (DHA, 22:6). Several
studies have shown that rebalancing the input of fatty acids by eating foods containing
omega-3 improves many biological parameters, such as triglyceride level, blood pressure and
cancer prevention (Burr, 2000; Kris-Etherton, Harris, Appel, & Committee, 2002;
Kryzhanovskii & Vititnova, 2009).
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However, highly unsaturated lipids (LH) are prone to oxidation, which takes place in
three main steps. The first one is the initiation step and consists of the loss of a hydrogen atom
under the action of trace metals, light or heat (Eq. 1). The resulting lipid radical (L•) reacts
with oxygen in a second step to form a peroxyl radical LOO• (Eq. 2). During the propagation,
LOO• radicals react with LH to form fatty acid hydroperoxides (LOOH), which are the
primary products of autoxidation (Eqs. 3 to 5). In a third step, i.e. the termination step, two
radicals react together to form non-radical products (Eqs. 6 to 8) (Kanner & Rosenthal, 1992).
Because of their large number of double bonds, omega-3 fatty acids undergo oxidative
degradation (Frankel, 1984; Shahidi & Zhong, 2010) leading to the modification of
organoleptic properties (Choe & Min, 2006) and the formation of lipid peroxides in food.
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To prevent omega-3 fatty acid from this oxidative degradation, phenols are widely
used as antioxidants. Phenolic compounds are the most frequently encountered antioxidants in
food. They significantly delay the oxidation of easily oxidizable materials, such as fats
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(Becker, Nissen, & Skibsted, 2004). Different types of antioxidants are commonly used: the
primary or chain-breaking antioxidants, such as the well-known butylated hydroxytoluene
(BHT), which convert PUFAs (polyunsaturated fatty acids) radicals (ROO•) into
hydroperoxides (ROOH) and act as chelators of metal ions (Marteau, Nardello-Rataj, Favier,
& Aubry, 2013; Reische, Lillard, & Eitenmiller, 2008). Nevertheless, due to an increase in
safety concerns and a strong push to replace them by natural alternatives, synthetic
antioxidants are increasingly disputed. Recent research has focused on the isolation and
identification of effective naturally occurring antioxidants, such as ascorbic acid (vitamin C)
and α-tocopherol (vitamin E).
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We report in this paper the study of 22 natural phenolic antioxidants in comparison to
7 synthetic ones using experimental and theoretical methods. First of all, bond dissociation
enthalpies (BDE) of the phenolic bond (O-H) have been calculated using the density
functional theory (DFT) method, providing a predictive scale of the antioxidant activity of the
phenols. These results have been correlated with the rate constants of hydrogen transfer from
phenolic antioxidants to the stable 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•).
Furthermore, measurements of oxygen consumption during the autoxidation reactions of
FAMEs linseed oil were carried out under oxygen pressure. This method allows the
determination of the induction periods (IP) and oxidation rates (Rox) of unsaturated oils in the
presence of phenolic antioxidants, reflecting their efficiency towards the preservation of
omega-3 oils. Very good correlations were obtained between the different approaches thus
leading to highlighting myricetin, rosmarinic and carnosic acids as efficient natural alternative
antioxidants to α-tocopherol.
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2. Materials and methods
2.1. Materials
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Solvents were of the purest grade commercially available from Sigma-Aldrich. The
2,2-diphenyl-1-picrylhydrazyl (DPPH•) radical was purchased from Sigma-Aldrich and kept
at a temperature lower than 5 °C. Aluminum oxide, basic, Brockmann I, for chromatography,
50-200 μm, 60 Å was from Acros Organics. Refined linseed oil was from the Vandeputte
Group, Belgium. FAME mix GLC-10 containing palmitic acid methyl ester (C16:0), stearic
acid methyl ester (C18:0), oleic acid methyl ester (C18:1), linoleic acid methyl ester (C18:2)
and linolenic acid methyl ester (C18:3) was from Supelco. Phenols 1 to 29 were purchased at
the purest grade from Sigma-Aldrich, Alfa Aesar, Cayman Chemical Company, ACROS,
Merck, TCI and Chromadex. For further details, see the supporting information.
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2.2. Calculation of bond dissociation enthalpies BDEs (O-H)
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Bond dissociation enthalpy (BDE) is given by the difference between the enthalpy of
the phenoxyl radical (plus that of the hydrogen atom) and that of the starting phenol as
described by Eqs. 9 and 10. All of the calculations were performed using Gaussian 03
packages (Szymusiak & Zielinski, 2003) as described in our previous work (Marteau,
Guitard, Penverne, Favier, Nardello-Rataj, & Aubry, 2016; Marteau, Nardello-Rataj, Favier,
& Aubry, 2013). The method is described as B3LYP/6-311++G(2d,2p)//B3LYP/6-311G(d,p).
Details are given in the supporting information.
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2.3. Determination of the rate constants for hydrogen transfer from phenols to DPPH• radical
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The analytic method to determine the kinetic rates of hydrogen transfer from phenols
to DPPH• radical (Eq. 11) has already been described in our previous works (Marteau,
Guitard, Penverne, Favier, Nardello-Rataj, & Aubry, 2016; Marteau, Nardello-Rataj, Favier,
& Aubry, 2013).
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The rate constants of hydrogen transfer k were determined by mixing the most reactive
phenols with DPPH• in stoichiometric amount. Under these conditions, second order kinetics
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(SOK) can be described by Eqs. 12 to 14. On the other hand, phenols with low reactivity, i.e.
4, 5, 10, 11 and 18, were introduced in excess at different concentrations. Under these
conditions, [ArOH] ≈ [ArOH]0 and pseudo first order kinetics (FOK) is described by Eq. 15.
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At 515 nm, DPPHꢀ and DPPH-H exhibit a strong (ε) and weak (ε’) molar absorption
coefficient, respectively, which allows the monitoring of the disappearance of DPPHꢀ (Eq.
11). Eqs. 14 and 15 can be expressed as a function of the absorbances A, A0 and Af (Eqs. 16
and 17), which provide the rate constants k and k[ArOH]0 as the slope of the linearized curve
obtained by plotting 1/(A – Af) and Ln(A – Af) / (A0 – Af) respectively versus time. In the
latter case, the experiment was performed at different concentrations of ArOH in order to
determine k.
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Fig. S2 shows an example of SOK reaction with α-tocopherol 1 in toluene. The rate constant
of the reaction was determined during the first 15 seconds of the reaction (Eq. 13) when the
reaction of the phenolic hydrogen with DPPH• occurs. The kinetic analysis holds as long as
aryloxyl radicals recombine instead of reacting with another DPPH• radical leading to ortho-
quinone derivatives in the case of catechol compounds. Fig. S3 shows an example of FOK
reaction with BHT 4 in toluene. To confirm that pseudo first order kinetic (FOK) and second
order kinetic (SOK) give equivalent results for the same phenolic antioxidants, vanillic acid
13 and eugenol 18 were studied under both conditions leading to similar values. Rate
constants k measured for phenols 1 - 29 are summarized in Table 2. For more information, see
the Supporting Information. Values of the kinetic rates of hydrogen transfer are given in the
Supporting Information (see Table S3).
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2.4. Determination of the stoichiometric number
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The stoichiometric number σexp is determined according to Eq. 18. The experiment
consists in measuring the final Af and initial A0 absorbances of a solution containing a large
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excess of DPPH• (1.5 x 10-4 M) compared to phenols (2.0 x 10-3 M). Values of σexp are
summarized in Table 2. For more details, see the supporting information (Table S5).
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2.5. Synthesis of antioxidant-free fatty acid methyl esters (FAMEs) by transesterification
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Linseed oil was purified 3 times beforehand by alumina column chromatography to
eliminate the antioxidants naturally present in neat oil. The transesterification reaction of
triglycerides of purified linseed oil with methanol into fatty acid methyl esters (FAMEs) is
given by eq. 19. 1 l of methanol was introduced into a 2l three-necked flask, equipped with a
condenser and a gas bubbler. Sodium (10 g, 2 equiv) was introduced piece by piece under
argon followed by purified linseed oil (200 g, 1 equiv). The reaction was performed during 12
hours under magnetic stirring. FAMEs were extracted with 3 x 300 ml of petroleum ether.
The combined organic phases were evaporated under pressure. Isolated FAMEs were stored
at -20 °C. The FAMEs were composed of 5.3 % of methyl palmitate (C16:0), 33.1 % of
methyl oleate (C18:1), 11.2 % of methyl linoleate (C18:2), 45.1 % of methyl linolenate
(C18:3, ω-3) and 5.3 % of methyl stearate (C18:0) as determined by GC/MS analyses.
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2.6. Analysis
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A Thermofisher GC Trace equipped with an AI 3000 injector connected to a
Thermofisher DSQ II simple quadrupole detector was used for the GC-MS analysis of
FAMEs. Compound separation was achieved on a 30 m, DB5MS with 0.25 mm i.d. and 0.25
µm film thickness gas chromatographic column (J&W Scientific, Folsom, CA, USA). Carrier
gas (ultra-pure helium) flow rate was 1.0 ml/min and the injector, the transfer line and the ion
source were maintained at 250, 270 and 220 °C, respectively. The MS detector was used in
the EI mode with an ionization voltage of 70 eV. The column was held at 130 °C for 0.5 min
and then programmed at 0.3 °C.min-1 to 180 °C and maintained for 5 min. Then, the column
was heated to 250 °C at 3 °C.min-1 and maintained for 10 min. The compounds were injected
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in the Split mode at a ratio of 20. FAME mix GLC-10 was used to analyze and quantify the
FAMEs composition.
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2.7. Autoxidation of FAMEs linseed oil in the presence of the phenolic antioxidants 1 to 29
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The autoxidation of FAMEs in the presence of the phenolic antioxidants was
investigated with the RapidOxy apparatus (Sch. 1) (Marteau, Ruyffelaere, Aubry, Penverne,
Favier, & Nardello-Rataj, 2013). 2 ml of FAMEs linseed oil were introduced into the
RapidOxy cell and 100 µl of phenol (1 to 29) solutions (10-2 mol.l-1) were then added to reach
a final antioxidant concentration of 5 x 10-4 mol.l-1. Antioxidants were solubilized in ethyl
acetate and an ultrasound bath was used to homogenize solutions. Small amounts of ethanol
could be used to pre-solubilize antioxidants.
As reported previously, the RapidOxy is particularly suitable to follow the inhibition
of oils autoxidation (Marteau, Ruyffelaere, Aubry, Penverne, Favier, & Nardello-Rataj,
2013). As shown in Figure 1, the curves corresponding to the oxygen consumption as a
function of time exhibit three stages: 1) after the set pressure (450 kPa) is reached, the cell is
heated to 90 °C and the pressure increases, 2) the maximum pressure stabilizes and then
slowly decreases during the induction period in which the phenolic antioxidant is effective
and the oxidation of FAMEs is prevented and 3) the phenol is completely consumed and the
oxidation of the oil takes place.
Moreover, the oxygen consumed as a function of time, i.e. -d[O2]/dt, can be used as an
indicator of the oxidability (Marteau, Ruyffelaere, Aubry, Penverne, Favier, & Nardello-
Rataj, 2013). Experiments were stopped when the pressure of oxygen had decreased to half of
the starting value after heating (Pf = Pmax/2). The evolution of the pressure was then converted
to a concentration of oxygen consumed in the liquid phase ∆[O2]t defined by Eq. 20 where
Vtot and Vliq are the volumes of the cell and of the FAMEs respectively, Pmax is the maximum
pressure obtained a few minutes after heating the cell and Pt is the pressure measured at a
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given time. The oxidation rate Rox is the rate of oxygen consumption and corresponds to the
slope of the trend curve of [O2] consumed during the second stage. Values of induction
periods (IP) and oxidation rates (Rox) are summarized in Table 2 and detailed in the
Supporting Information (Table S4).
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3. Results and discussion
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Antioxidants are of interest to the food industry because they prevent rancidity
(Halliwell, Aeschbach, Löliger, & Aruoma, 1995). To have a large overview of the different
classes of phenolic antioxidants, 29 phenols have been investigated (Scheme 2). α-
Tocopherol 1, chosen as the reference, is a very effective natural antioxidant widely used in
food. The ubiquitous synthetic antioxidants BHA 2, propyl gallate (PG) 3, BHT 4, o-tert-
butyl-p-cresol 5, TBHQ 6, 5-tert-butyl-pyrogallol 7 and 4-tert-butyl-catechol 8 have also been
studied for comparison.
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Gallic acid 9, protocatechuic acid 10, p-hydroxybenzoic acid (PHBA) 11, syringic acid
12 and vanillic acid 13 were selected as derivatives of hydroxybenzoic acid. In the same way,
caffeic acid 14, ferulic acid 15, chlorogenic acid 16 and sinapic acid 17 were retained because
they derive from hydroxycinnamic acid. Eugenol 18 and isoeugenol 19 are considered as
classical monophenols, with both fragrant and antioxidant properties, and were included to
investigate the effect of double bond position. Hydroxytyrosol 20 and catechol 21 are
polyphenols found in olive oil (Garcia Aranzazu, Ruiz-Mendez, Romero, & Brenes, 2006).
The other antioxidants represent the different classes of phenolic compounds: quercetin 22
and myricetin 23 for flavonoids (Pietta, 2000), resveratrol 24, aesculetin 25 and sesamol 26
are categorized as stilbenes, coumarins and lignans respectively. Finally, rosmarinic acid 27,
carnosic acid 28 and carnosol 29 are the major components of rosemary extract (Rosmarinus
officinalis L.) and are authorized in food in the form of extract (Erkan, Ayranci, & Ayranci,
2008).
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3.1. Phenolic antioxidants and bond dissociation enthalpy BDE (O-H)
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The activity of phenolic antioxidants is related to their ability to quench peroxyl
radicals by abstracting the phenolic hydrogen in a first step. An important parameter for
estimating antioxidant activity of phenol is the bond dissociation enthalpy (BDE) of the ArO-
H bond. A number of theoretical investigations with various basis sets (Bakalbassis,
Lithoxoidou, & Vafiadis, 2003; Klein & Lukes, 2006; Leopoldini, Russo, & Toscano, 2010;
Li, Liu, Fu, & Guo, 2007; Perez-Gonzalez, Rebollar-Zepeda, Leon-Carmona, & Galano,
2012) as well as experimental studies (Amorati & Valgimigli, 2012; Lucarini, Pedrielli,
Pedulli, Cabiddu, & Fattuoni, 1996; Lucarini & Pedulli, 2010) have been reported for the
determination of the BDE of phenols. It is noteworthy that theoretical results are basis set and
solvent dependent (Marteau, Guitard, Penverne, Favier, Nardello-Rataj, & Aubry, 2016).
Moreover, a systematic underestimation of about 5 kcal mol-1 can arise from the DFT
calculation. Hence, ∆BDE values compared to the theoretical BDE of phenol (82.2 kcal.mol-
1) should be considered (Tables 1, 2 and S1). The absolute BDE of phenols have been
calculated using the DFT method. The values concerning the group contribution of BDE are
consistent with published theoretical values as reported in Table 1 except for the methoxy
substituent (Table 1, entry 16) which is overestimated but can be discussed and explained.
Moreover, ∆BDE values of alkyl additive effect are consistent with the literature (Table S1).
All the calculated BDE values (Table 2) are in agreement with theoretical and experimental
values found in the literature.
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The effect of electron-donating and electron-withdrawing substituents has been
studied in numerous experimental and theoretical works from the early 90's (Lucarini &
Pedulli, 2010; Wright, Johnson, & DiLabio, 2001). In previous work, we also showed that
electron-donating groups decrease BDE values, in particular at the ortho- and para-positions
(Marteau, Nardello-Rataj, Favier, & Aubry, 2013). Here, we have extended the study by
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calculating using the same DFT method (described as B3LYP/6-311++G(2d,2p)//B3LYP/6-
311G(d,p)), the BDE of the ArO-H bond of 25 phenols bearing different substituents at the
ortho-, meta- or para- positions frequently encountered in natural phenols. Table 1
summarized the effect of substituents on hydrogen bonding and BDEs. The BDE values were
strongly dependent on the nature of the substituents, which were classified into four groups:
alkyl (-R), carboxyl (-CO2R), alkoxy (-OR) and hydroxyl (-OH) groups. The additive effect of
alkyl (CH3 and tert-butyl) groups has also been investigated (Table S1).
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Generally speaking, lower BDE values are obtained for electron-donating groups
(EDG) because they destabilize the phenol and/or stabilize the phenoxyl radical through
electronic effects and intramolecular hydrogen bonding (Lucarini, Pedrielli, Pedulli, Cabiddu,
& Fattuoni, 1996). As expected, alkyl substituents decrease the BDE via electronic effects
(Table 1). Ortho-methyl (entry 2), para-tert-butyl (entry 10), ortho-ethyl (entry 5) and ortho-
propenyl (entry 12) substituents decrease the BDE values of -2.0, -1.7, -2.1 and -3.0 kcal.mol-
1
respectively. Ortho-substitution by an OH group leads to a stronger decrease of the BDE
compared to a OCH3 group (entry 18 versus entry 15) (respectively -8.8 and -1.8 kcal.mol-1).
As reported by Wright and co-workers, this results from the disappearance of the
intramolecular hydrogen bonding in the phenoxyl radical in the case of OCH3 while with the
OH group, the radical stabilization becomes more important, besides the electronic effects of
the o-OH group, leading to a dramatic decrease of BDE (Wright, Johnson, & DiLabio, 2001).
Compared to the o-ally group (entry 11), the o-propenyl substituent (entry 12) allows further
electron delocalization, which increases the stability of the phenoxyl radical and leads to a
stronger BDE decrease from -3.0 and -2.0 kcal.mol-1 respectively. However, meta- alkyl,
alkoxy and hydroxyl groups have low or no impact compared to ortho- and para-
substitutions (Chandra & Uchimaru, 2002; Lucarini & Pedulli, 2010; H. Y. Zhang, Sun, &
Chen, 2001).
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On the contrary, electron withdrawing groups (EWG) induce a stabilization of the
phenol and/or a stabilization of the phenoxyl radical (Lucarini, Pedrielli, Pedulli, Cabiddu, &
Fattuoni, 1996) leading thus to higher BDEs. As an example, a -COOH group in the ortho-
position allows intramolecular hydrogen bonding, which increases the stabilization of the
phenol (Korth, de Heer, & Mulder, 2002; Wright, Johnson, & DiLabio, 2001). Nevertheless,
it disappears when the phenoxyl radical is formed. Indeed, both effects are unfavorable for an
easy transfer of the hydrogen involving a huge increase of +13.0 kcal.mol-1 (entry 21).
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Using ReqEPR, Pedulli and co-workers have demonstrated the additive effect of some
alkyl substituents and supported the results with some additivity rules (Lucarini & Pedulli,
2010). According to theoretical calculation using the B3LYP/6-311++G(2d,2p)//B3LYP/6-
311G(d,p) DFT method, additive effects are also found for methyl and tert-butyl substituents
(Table S1).
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In order to establish a scale of the antioxidant activity, the B3LYP/6-
311++G(2d,2p)//B3LYP/6-311G(d,p) DFT method has been applied to the 29 phenolic
antioxidants (Scheme 3). As mentioned previously, the values of the BDEs are strongly
influenced by the number, nature and position of the substituents linked to the phenol ring.
According to the BDE values and the chemical structures of the phenols, three main classes of
antioxidants can be distinguished: 1) those with very low BDE (from 66 to 72 kcal.mol-1), 2)
those with medium BDE (from 72 to 79 kcal.mol-1) and 3) those with high BDE (from 79 to
86 kcal.mol-1). This division is arbitrary but allows the rationalization of the structural effects
in each set.
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Very low BDEs have been found for 5-tert-butylpyrogallol 7, myricetin 23, propyl
gallate 3 and gallic acid 9. Wright and co-workers have reported that the central phenoxyl
radical stabilized by two hydrogen bonds would be the more stable conformer compared to
the phenol stabilized by only one intramolecular hydrogen bonding (Eq. 21) (Wright,
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Johnson, & DiLabio, 2001). As the phenol parent is the same in both cases, BDE is lower
when the phenoxyl radical is formed at the central hydroxyl group (see Table S2). This class
also contains α-tocopherol 1 with a BDE of 69.1 kcal.mol-1. Such a low BDE results from
different factors: 1) the alkoxyl group in the p-position, 2) the four alkyl substituents on the
phenolic ring and 3) the molecular rigidity imposed by the pyran structure (Marteau,
Nardello-Rataj, Favier, & Aubry, 2013). Finally, quercetin 22, rosmarinic acid 27, carnosic
acid 28 and carnosol 29 also exhibit very low BDEs. Indeed, they bear a catechol-type ring
moiety, conjugated double bonds and alkyl substituents on the phenol rings which strongly
contribute to lower the BDE.
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On the other hand, catechol 21 itself and catechol-based derivatives with COOH
groups (i.e. protocatechuic acid 10, caffeic acid 14), OCH3 groups (i.e. BHA 2, syringic acid
12, sinapic acid 19) and conjugated double bonds (i.e. chlorogenic acid 16, aesculetin 25)
have medium BDEs as well as those with different alkyl substituents (i.e. BHT 4, o-tert-butyl-
p-cresol 5, 4-tert-butyl-catechol 8) and substituted hydroquinone (i.e. TBHQ 6).
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Finally, ferulic acid 15, eugenol 18, vanillic acid 13 and PHBA 11 have high BDEs.
Eugenol 18 has a higher BDE than isoeugenol 19 because of the non-conjugated double bond.
Vanillic acid 13 and PHBA 11 have higher BDEs than phenol itself. This is due to the effect
of the electron withdrawing carboxyl group in the para position (Table 1, entry 23), which
results in an increase of +2.5 kcal.mol-1. This effect is not fully balanced by the ortho-OCH3
group (Table 1, entry 15), which allows a decrease of -1.8 kcal.mol-1.
290
291
292
293
Based on these theoretical calculations, gallic acid 9 as a derivative of hydroxybenzoic
acid, myricetin 23 as a flavonoid and rosmarinic acid 27, carnosic acid 28 and carnosol 29 as
phenols present in rosemary extracts are expected to be very effective antioxidants towards
omega-3 oils autoxidation as they have a very low BDE.
294
3.2. Phenolic antioxidants and kinetic rates of hydrogen abstraction (DPPH• test)
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297
298
299
300
301
302
303
304
305
To confirm the antioxidant activity of phenols highlighted above on the basis of
thermodynamic data, rate constants of hydrogen transfer have then been determined using the
DPPH• (2,2-diphenyl-1-picrylhydrazyl) test (Sharma & Bhat, 2009). The DPPH• radical is
stable at room temperature and its disappearance can be readily followed by UV-visible
spectroscopy. Thus, rate constants of hydrogen transfer from the phenols to DPPH• were
obtained using pseudo-first order (FOK) and second order (SOK) kinetics. Toluene was
chosen as an inert solvent. The mechanism involved in this apolar and aprotic solvent is called
hydrogen atom transfer (HAT) in contrast to the sequential proton loss electron transfer
(SPLET) mechanism which takes place in polar solvents (Amorati, Pedulli, Cabrini,
Zambonin, & Landi, 2006; Litwinienko & Ingold, 2005; Lucarini & Pedulli, 2010; Marteau,
Nardello-Rataj, Favier, & Aubry, 2013).
306
307
308
309
310
311
312
313
314
315
316
317
318
319
As expected, the logarithm of the rate constants for the reaction of hydrogen transfer is
well correlated with the calculated BDEs of phenols (R² = 0.95) confirming that the HAT
mechanism occurs in toluene (Fig. 2) (Foti, Daquino, Mackie, DiLabio, & Ingold, 2008; Foti,
Johnson, Vinqvist, Wright, Barclay, & Ingold, 2002; Marteau, Nardello-Rataj, Favier, &
Aubry, 2013). Nevertheless, there are some exceptions. Indeed, due to steric hindrance of
ortho- and di-ortho-tertbutyl groups such as in BHA 2, o-tert-butyl-p-cresol 5 and BHT 4, the
kinetic transfer of the phenolic hydrogen is sharply slowed down as explained by Marteau et
al. (Marteau, Nardello-Rataj, Favier, & Aubry, 2013). Concerning the natural phenolic
antioxidants, α-tocopherol 1 exhibits the higher rates (2670 M-1.s-1) followed by carnosol 29
(1680 M-1.s-1), hydroxytyrosol 20 (1070 M-1.s-1) and carnosic acid 28 (640 M-1.s-1). Log k
decreases when the BDE values increase as also demonstrated by Foti et al. in heptane (Foti,
Daquino, Mackie, DiLabio, & Ingold, 2008) and Marteau et al. in m-xylene (Marteau,
Nardello-Rataj, Favier, & Aubry, 2013). Foti and co-workers have also proven that the
kinetics obtained with the DPPH• method are correlated with the reaction of phenols with
13

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323
peroxyl radicals (LOO•). Indeed, potent chain-breaking antioxidants react with LOO• and not
with L• because its reaction with O2 is too fast. Accordingly, this test can mimic the behaviour
of phenolic antioxidants during the inhibition of oils oxidation thanks to hydrogen transfer
with a radical mechanism (Foti, Johnson, Vinqvist, Wright, Barclay, & Ingold, 2002).
324
3.3. Stoichiometric number (σ) of phenols 1 to 29
325
326
327
328
329
330
331
332
Phenolic antioxidants are also characterized by the maximum number of radicals
trapped by one antioxidant molecule, named the stoichiometric number σexp. It is determined
with the DPPH• test from the final absorbance reached by DPPH• in the presence of a large
excess of DPPH• with respect to the antioxidant concentration (Eq. 18) (Goupy, Dufour,
Loonis, & Dangles, 2003). All of the stoichiometric numbers σexp determined for phenols (see
Table S5 in the Supporting Information) are summarized in Table 2. Toluene was replaced by
ethyl acetate when antioxidants were not soluble. Figure S4 reports the results for α-
tocopherol 1 at various concentrations.
333
334
335
336
337
338
339
The stoichiometric number depends on the nature of the substituents at the ortho and
para positions. The determination of the number of hydrogen transfer provides some
information on the oxidation products, but, usually, several competitive pathways are
simultaneously involved, complicating the interpretation. The mechanism of interaction
between the DPPH• radical and the phenol takes place in two steps: 1) abstraction of the
phenolic hydrogen and 2) transfer of a second hydrogen or formation of reactive or non-
reactive dimers from the phenoxyl radical ArO•.
340
341
342
343
Phenolic antioxidants with catechol structure 30 are known to transfer 2 hydrogens to
DPPH•. Indeed, the phenolic hydrogen from the ortho-OH substituent releases its second
hydrogen and the ortho-quinone compound 31 is formed (Eq. 22). The global stoichiometric
numbers of 2.0 are in agreement with the reported oxidized forms of catechol derivatives 7
14

344
345
346
347
348
349
350
351
352
353
(Tkachev, Aldoshin, Shilov, Komissarov, Sayapin, Korobov, et al., 2007), 10 (Saito &
Kawabata, 2006), 14 (Arakawa, Yamaguchi, Hotta, Osakai, & Kimoto, 2004), 16 (Pierpoint,
1966) , 20 (De Lucia, Panzella, Pezzella, Napolitano, & D'Ischia, 2006), 21 (Balla, Kiss, &
Jameson, 1992), 22 (McPhail, Hartley, Gardner, & Duthie, 2003), 28 (Masuda, Inaba, &
Takeda, 2001) and 29 (Masuda, Kirikihira, & Takeda, 2005). With 2 active catechol moieties
(BDEs of 69.2, 72.4, 75.2 and 75.9 kcal.mol-1), rosmarinic acid 27 traps 4 radicals per
molecule and forms 2 ortho-quinone substructures 32 in the same molecule (Fujimoto &
Masuda, 2012), which correlates with a stoichiometric number of 4.1 (Eq. 23). Antioxidants
with hydroquinone structure such as 6 give a stoichiometric number of 2.0 due to the
formation of p-quinone compound 33 (Eq. 24) (Hamama & Nawar, 1991).
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
Surprisingly, gallic acid 9, propyl gallate 3, myricetin 23 and 4-tert-butylcatechol 8
trap, respectively, 5.0, 3.9, 3.4 and 2.5 radicals per molecule. Given the number of phenolic
hydrogens in their chemical structure, it is assumed that they form active dimers which react
again with a DPPH• radical leading to an increase of the stoichiometric number and the
formation of oxidized antioxidant dimers (Eq. 25) as already observed. (Arab Chamjangali,
Bakherad, & Ameri, 2015; Caregnato, Gara, Bosio, Gonzalez, Russo, Michelini, et al., 2008;
Imagawa & Takino, 1962).
Monophenols, which can apparently trap only one hydrogen per molecule, are in fact
able to transfer more than one. As already published in our previous work, α-tocopherol 1
(Eq. 26) and BHT 4 (Eq. 27) firstly transfer the phenolic hydrogen and then a second
hydrogen from the o- or p- methyl group leading to a o- or p- quinone-methide derivative,
which is consistent with a stoichiometric number σexp of 2. These p- and o- quinone-methide
derivatives 36 and 38 react with starting phenols 1 and 4 to form dimers 37 and 39.
Nevertheless, quinone-methides react very slowly with starting phenols and dimers are not
obtained (Marteau, Guitard, Penverne, Favier, Nardello-Rataj, & Aubry, 2016).
15

369
370
371
372
373
374
375
376
377
378
379
380
381
Moreover, o-tert-butyl-p-cresol 5 traps 2.5 radicals per molecule via 2 competitive
pathways: 1) formation of p-quinone methide 41 (Eq. 29) and 2) formation of dimer 42 via an
ortho-dimerization (Eq. 30) (Marteau, Guitard, Penverne, Favier, Nardello-Rataj, & Aubry,
2016).
BHA 2, ferulic acid 15 and eugenol 18 with free ortho positions dimerize at the ortho-
ortho positions giving diphenols 40 with strong antioxidant activity since they can further
transfer two hydrogen atoms (Eq. 28) (Bortolomeazzi, Verardo, Liessi, & Callea, 2009;
Carunchio, Crescenzi, Girelli, Messina, & Tarola, 2001; Kadoma, Ito, Yokoe, & Fujisawa,
2008; Omura, 2008). Sesamol 26 releases its first phenolic hydrogen and oxidized compound
43 is formed via dimerization (Constantin, Conrad, & Beifuss, 2012). It could then transfer 2
more hydrogens, which would be in agreement with a stoichiometric number of 2.1 (Eq. 31).
Some monophenols, such as isoeugenol 19, trap only one radical per molecule because they
form non-reactive dimers 44 (Eq. 32) (Bortolomeazzi, Verardo, Liessi, & Callea, 2009).
382
383
384
385
386
387
388
The determination of the stoichiometric number σexp highlights that catechol and
pyrogallol structures trap the highest number of radicals per molecule. The formation of
active dimers, via C-C and C-O couplings, improves the number of hydrogens released by a
molecule. Compared to α-tocopherol 1 with a stoichiometric number of 2, gallic acid 9,
myricetin 23 and rosmarinic acid 27 trap 5.0, 3.4 and 4.1 radicals, respectively. This
parameter could play an important role regarding the antioxidant capacity of phenols during
the oxidation of omega-3 oils.
389
3.4. Phenolic antioxidants and inhibition of FAMEs linseed oil oxidation (RapidOxy test)
390
391
392
Finally, besides the BDE calculations and the DPPH• test, the autoxidation of omega-3
oils in the presence of phenols has been kinetically investigated by measuring the oxygen
consumption using the RapidOxy apparatus, which provides information on the induction
16

393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
periods (IP) and oxidation rates (Rox). Autoxidation of oils and more precisely edible oils rich
in omega-3 by oxygen (3O2) has been known for a long time (Frankel, 1984). The oxidation
mechanism of methyl linolenate, the major omega-3 fatty acid methyl ester (FAME), starts
with the abstraction of the bis-allylic hydrogens on C11 and C14 by the way of heating and UV,
producing two pentadienyl radicals as primary radicals; this takes place during the initiation
step. Then O2 attacks at the terminal position of these radicals and produces a mixture of 9-,
12-, 13- and 16- conjugated diene-triene hydroperoxides isomers providing primary oxidation
products. GC-MS (Chan, 1977) and HPLC (Neff, Frankel, & Weisleder, 1981) analyses have
shown that there is a significantly higher proportion of the outer 9- and 16-hydroperoxides
than of the internal 12- and 13- hydroperoxides. This uneven distribution of isomeric
hydroperoxides is due to the 1,3- and 1,4-cyclisations of the internal 12- and 13-hydroperoxyl
radicals into hydroperoxy cyclic peroxides (Neff, Frankel, & Weisleder, 1981) leading to
secondary oxidation products. The 1,3-cyclisation is also accompanied by a second
cyclisation to form bicycloendoperoxides structurally related to prostaglandins. These
different steps are summarized in Scheme S1.
408
409
410
411
412
413
414
415
416
Phenolic antioxidants are usually incorporated into edible oils rich in omega-3 to avoid
their oxidative degradation. Their most labile hydrogen reacts with radicals and converts them
into more stable non radical products (Decker, 2002). As reported by Choe et al., antioxidants
with a reduction potential (500 mV or below) lower than that of alkoxyl, peroxyl and alkyl
radicals of polyunsaturated fatty acids (respectively 1600, 1000 and 600 mV) can transfer
hydrogen to a radical (Choe & Min, 2006). This clearly shows that antioxidants react with
lipid peroxyl radicals before they react with other lipid molecules to produce further free
radicals as shown in Scheme S2. For the purpose of the current study, FAMEs of linseed oil
were synthesized by transesterification.
17

417
418
419
420
421
422
423
424
425
Induction periods (IP) and oxidation rates (Rox) obtained for the 29 phenolic
antioxidants are summarized in Table 2. Three categories of phenolic antioxidants can be
distinguished considering the induction period (IP) as shown by Figure 3. First, phenols
included in category A (7, 8, 23, 27 and 28) have an IP higher than 200 min and are highly
effective antioxidants. Then, phenols of category B (1, 2, 3, 4, 9, 14, 16, 20, 21, 22, 25, 26 and
29) have an IP between 200 and 100 min and are considered as moderately effective
antioxidants. Finally, phenols 5, 6, 10, 11, 12, 13, 15, 17, 18, 19 and 24 belong to category C
and have an induction period lower than 100 min. They are considered as poorly effective
antioxidants.
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
Figure 3 reports the stoichiometric numbers obtained with the DPPH• test as a function
of the induction periods for the different antioxidants. The trend is clear: highly effective
antioxidants are polyphenols with high stoichiometric numbers. Indeed, the number of
hydrogens released by one molecule of antioxidant has an important impact on the inhibition
of oxidation and influences the induction period. Thus, myricetin 23, rosmarinic acid 27 and
4-tert-butylcatechol 8 have stoichiometric numbers higher than 2.0 and are more efficient
than α-tocopherol 1. However, 5-tert-butylpyrogallol 7 and carnosic acid 28 are also more
efficient than α-tocopherol 1 while they trap the same number of radicals. Moderately
effective antioxidants such as gallic acid 9, propyl gallate 3, hydroxytyrosol 20, carnosol 29
and caffeic acid 14 exhibit interesting capacity against FAMEs oxidation and have almost the
same effectiveness as α-tocopherol 1. Basically, they trap 2 radicals except for gallic acid 9
and propyl gallate 3 which have higher stoichiometric numbers. It is noteworthy that carnosol
29 is less efficient than carnosic acid 28 while they have a similar chemical structure and
equal stoichiometric number (σexp = 2.0). This is probably due to the fact that carnosic acid 28
is the starting phenol and carnosol is obtained during its degradation pathway as shown by
Zhang et al. (Y. Zhang, Smuts, Dodbiba, Rangarajan, Lang, & Armstrong, 2012). Finally,
18

442
443
444
445
446
poorly effective antioxidants such as sinapic acid 17, isoeugenol 19 and resveratrol 24 exhibit
the lowest induction periods and stoichiometric numbers. They do not transfer enough
hydrogen to be efficient at delaying action of the oxidation process. Nevertheless, o-tert-
butyl-p-cresol 5, protocatechuic acid 10 and eugenol 18 trap 2 or more hydrogens without
impeding the oxidation of FAMEs.
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
Accordingly, the number of radicals trapped by a molecule of phenol (σexp) is a crucial
parameter for the protection of FAMEs against oxidation but the exceptions above-mentioned
point out other essential factors than just the BDE. Indeed, there is a very good correlation
between oxidation rates (Rox) and BDE values as shown in Fig. 4 (R² = 0.97). We have
previously shown a correlation between BDEs and rate constants k obtained with the DPPH•
test in toluene involving a transfer of hydrogen according to the HAT mechanism. Thereby,
we can assume here that the mechanism involved during the inhibition of oxidation by
phenolic antioxidants is a radical mechanism. During the oxidation of the FAMEs linseed oil,
the lower the oxygen consumption is, the more efficient the antioxidant is. Myricetin 23 with
a BDE of 67.4 kcal.mol-1 provides an oxidation rates of 0.11 mM-1.s-1 whereas vanillic acid
13 with a BDE of 83.14 kcal.mol-1 does not reduce the oxidation rate (1.03 mM-1.s-1).
Sterically hindered phenols (i.e. BHA 2, o-tert-butyl-p-cresol 5 and BHT 4) which did not
follow the trend curve with the DPPH• test are now included in the tendency. Gallic acid 9
and propyl gallate 3, identified as exceptions by their higher stoichiometric number (5 and 3.9
respectively), do not transfer their hydrogen fast enough and the oxidation takes place at a rate
of 0.32 and 0.26 mM.min-1. Therefore, even if antioxidants could transfer more than 2
hydrogens, they would not be efficient enough to avoid the autoxidation of omega-3 oils.
464
465
466
Consequently, the number of radicals trapped by the phenolic antioxidant (σexp) and
the BDE value are the two essential parameters depicting the antioxidant efficiency of
phenols towards oils autoxidation. Indeed, phenols with the highest stoichiometric number
19

467
468
and the lowest BDE are the more efficient antioxidants. Thus, myricetin 23, rosmarinic acid
27 and carnosic acid 28 are more effective than α-tocopherol 1.
469
470
471
472
473
474
475
476
The better antioxidant capacity of myricetin 23 compared to that of α -tocopherol 1
has already been reported with regards to other targets: triacylglycerols of sunflower oil
(Marinova, Toneva, & Yanishlieva, 2007), methyl esters of soybean oil (Moser, 2008) and
unilamellar liposomes (Gordon & Roedig-Penman, 1998). The better effectiveness of carnosic
acid 28 has been shown by the improvement of the stability of squalene under heating and UV
irradiation (Wang, Wang, Yang, Zu, Liu, & Liu, 2011). Finally, rosmarinic acid 27 has been
found to be more efficient than α-tocopherol 1 during the inhibition of lipid oxidation in
cooked ground beef (Alamed, Chaiyasit, McClements, & Decker, 2009).
477
478
479
480
481
482
Carnosol 29, quercetin 22, hydroxytyrosol 20 and caffeic acid 14 are as effective as α-
tocopherol 1. They all have a BDE lower than 72.1 kcal.mol-1 and a stoichiometric number
equal to 2. Apart from α-tocopherol 1, which is well known as an effective phenolic
antioxidant, myricetin 23, rosmarinic acid 27 and carnosic acid 28 are relevant alternatives to
synthetic antioxidants, such as propyl gallate 3, BHA 2 and BHT 4, which have similar or
higher oxidation rates.
483
4. Conclusion
484
485
486
487
488
489
490
Edible oils rich in omega-3 are very sensitive to autoxidation because of the large
number of double bonds in their molecular structure. Following a radical mechanism of
degradation with steps of initiation, propagation and termination, fatty acids are converted
into hydroperoxides as primary oxidation products. The preservation of these edible oils
against oxidation is essential to enable a longer product lifetime and maintain their
organoleptic properties. Many publications are related to the inhibition of triglycerides
oxidation by antioxidants but few of them are focused on omega-3 oils.
20

491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
Bond dissociation enthalpies (BDEs) calculated for 29 phenols have confirmed the
important role of electron-donating groups on the phenol ring, providing a theoretical
classification of these phenols regarding their antioxidant potential. Thus, low BDE values
were found for myricetin 23, rosmarinic acid 27 and gallic acid 9, highlighting their capacity
to act as efficient antioxidants. The pyrogallol and bis-catechol moieties are key structural
parameters to reach very low BDE values. On the other hand, the DPPH• test, which mimics
well the reaction of peroxyl radicals LOO• with phenolic antioxidants, has provided the rate
constants of hydrogen transfer from phenols to radicals. In non-polar solvents, the HAT
mechanism prevails and α-tocopherol 1, carnosic acid 28 and carnosol 29 are the best natural
phenolic antioxidants. The DPPH• test also provides useful information through the
determination of the stoichiometric number. The formation of dimers and trimers, via C-C
and C-O couplings, with an antioxidant activity higher than that of the starting phenol,
improves the number of hydrogens released by a molecule. With a stoichiometric number
higher than 2, gallic acid 9, myricetin 23 and rosmarinic acid 27, respectively, trap 5.0, 3.4
and 4.1 radicals per molecule. This key parameter reflects the antioxidant effectiveness of
phenols. Finally, monitoring the oxygen consumption during the autoxidation of FAMEs rich
in omega-3 has been shown to be well suited to study both the oxidative degradation of such
oils and their preservation.
509
510
511
512
513
514
515
Remarkably, very good correlations between the three approaches were found. The
correlation between oxidation rates and BDEs clearly shows that the mechanism involved
during the inhibition of oils oxidation by phenolic antioxidants is a radical mechanism.
Furthermore, the number of radicals trapped by molecules of phenols influence their
induction periods. Consequently, stoichiometric number and BDE govern the efficiency of
phenols. Thereby, the longer the induction period is, the better the effectiveness of phenolic
antioxidant against oxidation of FAMEs. Based on these findings, requirements for efficient
21

516
517
518
519
520
521
522
antioxidants for the preservation of omega-3 oils against oxidation are: 1) presence in the
structure of catechol or pyrogallol moieties, 2) low bond dissociation enthalpy (BDE), 3)
formation of active dimers during the oxidation process and 4) high number of hydrogens
released. Thus, with the best capacities to trap radicals per molecule combined with the lowest
BDE, myricetin 23, rosmarinic acid 27 and carnosic acid 28 are the best natural alternatives to
α-tocopherol 1 and synthetic antioxidants to avoid or at least slow down the autoxidation of
edible oils rich in omega-3 and sustain their physiological benefits.
523
Acknowledgements
524
525
526
Chevreul Institute (FR 2638), Ministère de l’Enseignement Supérieur et de la Recherche,
Région Nord – Pas de Calais, FEDER and the company Cargill are acknowledged for
supporting and partially funding this work.
527
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Equations
LH Initiator→ L• + H•
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
3
L• + O2 → LOO•
LOO• + LH → LOOH + L•
LOO• + C=C → LOOC−C•
LOOC−C• + O2 → LOOC−COO•
3
LOO• + LOO• → LOOOOL
LOO• + L• → LOOL
L• + L• → LL
ArO − H + X• → ArO• + X − H
BDE (ArO− H) = H0f (ArO• ) + Hf0 (H• ) − Hf0 (ArO− H)
(11)
(12)
d
[
DPPH•
]
= −k
[
DPPH•
]
[ArOH
]
dt
27

d
[
DPPH•
]
2
•
]
(13)
[
= − k DPPH
dt
1
1
−
= − k t
(14)
(15)
(16)
[DPPH• ] [DPPH• ]0
[
DPPH• DPPH•
]
=
[
]
exp
{
− k
[ArOH 0
] }
t
0
1
1
k
=
−
t
(A − Af )
(A0 − Af )
(ε − ε')
(A − Af )
Ln
= − k [ArOH]0 t
(17)
(18)
(A0 − Af )
[DPPH• ]0 −[DPPH• ]f
A0 − Af
σexp
=
=
[ArOH]0
(ε − ε')[ArOH]0
O
C16:0
C18:0
C18:1
O
O
O
O
O
O
O
O
OH
O
O
O
MeOH
(19)
OH
OH
+
O
O
Na (2eq)
Argon
O
C18:2
C18:3
O
(Vtot − Vliq )
(Pmax − Pt )
∆[O2 ]t =
×
(20)
(21)
RT
Vliq
(22)
28

(23)
(24)
H
R
O
OH
O
H
OH
2
2
-H
DPPH
+
2
+
DPPH
HO
OH
R
R
30
34
(25)
R
H
R
O
O
H
O
H
O
O
4
+
DPPH
4
-H
DPPH
+
O
O
H
O
R
R
34
35
R
O
O
H
37
1
O
O
H
(26)
2
H
R
+
DPPH-
2
+
DPPH
R
O
Very slow
reaction
O
O
R
O
1
36
(27)
(28)
29